Consumption of fruits and vegetables reduces the risk of several chronic diseases, most notably cardiovascular disease and cancer (Fung et al., Am J Clin Nutr 92:1429-1435, 2010). Although fruits and vegetables are important sources of vitamins and minerals, the salutary effects of these foods are attributed primarily to their content of fiber and antioxidants. Selected vitamins, such as vitamins C and E, exert antioxidant activity primarily through direct scavenging of oxygen and nitrogen radicals. Diverse phenolic substances (also called polyphenols) in fruits and vegetables also possess considerable antioxidant capacity and influence multiple cellular signaling cascades. Polyphenols are considered of key importance for mediating the health benefits of the Mediterranean diet and red wine (Del Rio et al., Antioxid Redox Signal 18:1818-1892, 2013).
Grapes are a particularly rich source of polyphenols, which is thought to explain the health benefits of consuming grapes, grape juice and wine. The predominant polyphenol species found in grapes (white grapes in particular) are similar to those identified in green tea and cocoa, namely flavanols or flavan-3-ols (Del Rio et al., Antioxid Redox Signal 18:1818-1892, 2013). Grape seeds are often considered a waste stream produced by the commercial production of grape juices, jellies, purees, and wine. The recognition that the seeds are the most concentrated source of polyphenols within the grape has prompted the development of a variety of industrial processes for grape seed processing to produce concentrated polyphenol extracts. Typically, these processes yield extracts containing the entire complement of phenolics found within the seed ranging from monomeric flavanols such as catechin and epicatechin to oligomers of two to ten of these monomeric units (known as procyanidins, proanthocyanidins, or oligomeric proanthocyanidins which are abbreviated as OPCs) to high molecular weight polymers of these monomeric units (known as polymeric procyanidins, tannins or condensed tannins). However, research over the last decade has revealed differing bioavailability and diverse biological actions of monomeric versus oligomeric versus polymeric catechin-based polyphenols. Unfortunately, most existing extraction processes for concentrating polyphenols employ harsh solvents that must be removed through complex fractionation schemes. Other fractionation methods use adsorption methodology that uses solvents such as ethanol for elution, necessitating specialized production facilities.
U.S. Pat. Nos. 3,435,407 and 4,698,360 disclose a method for the extraction of proanthocyanidins from pine bark, while minimizing the extraction of accompanying polymeric polyphenols (condensed tannins) using sodium chloride extraction and precipitation. These methods require repeated extraction with ethyl acetate. The ethyl acetate is then removed by evaporation, and oligomeric procyanidins are precipitated by the addition of chloroform.
U.S. Pat. No. 5,484,594 discloses a multistage process for preparing grape seed extract rich in oligomeric proanthocyanidins but that is almost free of monomers, which according to that patent are undesirable waste. The seeds are extracted with 80% aqueous acetone, filtered to remove polymeric waste product, then the solvent is evaporated, and the remaining polyphenols are concentrated. These steps are followed by further filtration to eliminate undesired monomers. The process uses chlorinated hydrocarbons, which are known carcinogens. The use of acetone and chlorinated hydrocarbons also increases the possibility of reactions with the procyandins and an alteration in the nature of the extracted procyanidins.
U.S. Pat. No. 5,912,363 discloses extraction of proanthocyanidins from plant material by heating an aqueous solid plant material mixture at temperatures of up to 350° F. under increased pressure and/or reduced oxygen, followed by membrane filtration to produce a permeate containing extracted proanthocyanidins. The extracted proanthocyanidins are then separated from the permeate by contacting the permeate with an adsorbent resin and subsequent elution with ethanol. All the examples are carried out at a temperature of 240° F., which is over the boiling point of water at atmospheric pressure. No selectivity for oligomers with low degrees of polymerization (dp) is disclosed.
U.S. Pat. No. 6,544,581 discloses a process for non-selectively extracting oligomeric and polymeric polyphenols from whole grapes, seeds or pomace using hot water extraction and a dual pH treatment. Oligomeric procyanidins having a dp up to 7 and polymeric procyanidins having a dp up to 16 are obtained in a process that uses mineral acids, such as sulfuric acid, then neutralized with an alkali metal base such as sodium or potassium hydroxide. The extract is treated with an adsorption resin and the polyphenols are eluted with ethanol. The resulting product has a very high level of total phenols, but relatively low percentages of monomeric procyanidins.
U.S. Pat. No. 7,767,235 produces a grape, grape seed or grape pomace extract by acidifying the extract with a mineral acid, such as sulfuric acid and then filtering it with diatomaceous earth. The extract is said to have about 5-15% monomeric procyanidins.
U.S. Pat. No. 8,075,929 produces a grape extract having about 5-15% monomers by extracting grapes at elevated temperatures, treating the extract with tannase, and acidifying the extract to a pH of about 1.5-2.5.
Prior procyanidin extraction processes generally extract grape seeds with biologically less desirable acidified organic solvents such as methanol, ethanol or acetone that must be removed prior to administration to a subject. Other prior extraction processes require high temperature processing. Some methods use pressurized liquid extraction (PLE) under high pressure and temperatures, successive chloroform in methanol extractions, or acidification with mineral acids such as sulfuric acids. Even with such potentially toxic treatments, the resulting extracts are not very enriched in monomeric procyanidins and have no more than about 15-17% monomers in the extract.
It would be desirable to provide a more biologically compatible process that is capable of extracting vegetable material, such as grape seeds, to obtain an isolated procyanidin extract having a low mean degree of polymerization (mdp). It would also be advantageous to provide such a method in which other biologically useful components of the grape seed were obtained and isolated, along with monomeric and dimeric flavanols.